Arg123 in the rat muscarinic M1 receptor is part of the highly conserved triad Asp-Arg-Tyr found at the junction of transmembrane helix 3 with the second intracellular loop. Mutation of Arg123 to Lys, Ala, Leu, Glu and Gln had no effect on levels of receptor expression in COS-7 cells, or on affinities for the antagonist N-methylscopolamine. Acetylcholine stimulation of the Lys123 receptor evoked the same maximum phosphoinositide response as the wild type, although the potency was reduced six-fold, but mutation to other residues strongly disrupted receptor function. Mutation of Arg123 always decreased the ratio of the high affinity to the low affinity agonist binding constant, but the absolute effect on the latter varied from a 4-fold increase for the Lys123 to a small decrease for the Leu123 mutation. These results suggest that a positive charge at position 123 is central to the activation of G-proteins by the muscarinic M1 receptor.